1. Field of the Invention
This invention relates to an assay for detecting the levels of free, circulating insulin-like growth factor-I or -II (IGF-I or -II) or growth hormone (GH) in a body fluid such as plasma or serum not bound to its respective binding protein(s).
2. Description of Related Disclosures
For diagnosing clinical disorders and making decisions regarding appropriate therapy, assay methods have been developed to detect levels of a particular protein indicative of the disorder in a body fluid sample. Since some proteins circulate in the body bound to carrier or binding proteins, assay methods are needed to detect the amount of circulating free protein to obtain an accurate diagnosis. For example, it is desirable to determine blood levels of free IGF-I and GH to diagnose for hypoglycemia, gigantism, acromegaly, hypopituitarism, pituitary dwarfism, GH deficiency, thymomegalia, and CNS diseases such as Alzheimer's disease.
Many assay techniques have been used to detect the somatomedin (IGF) activities of plasma. As is true of most hormones, the original techniques used were bioassays. Salmon and Daughaday, J. Lab and Clin. Med., 49: 825-836 (1957). While such techniques measure a biologic action, they are cumbersome to perform, show a great deal of intra-assay variability, have a relatively high detection limit, are subject to the effects of inhibitory substances, and lack specificity.
The availability of pure peptide for iodination permitted the development of radioreceptor assays (RRA) using tissues as the receptor source. Marshall et al., J. Clin Endocrinol. Metab., 39: 283 (1974); Megyesi et al., Biochem. Biophys. Res. Commun., 57: 307 (1974). A typical RRA protocol involves incubating 100-250 .mu.g of human placental membrane protein with tracer and standards or samples for 16 hours at 2.degree. C., then separating the bound and free ligand by centrifugation. The specificity of this assay is determined by the tracer peptide, but with either IGF-I or IGF-II tracer, there is some cross-reactivity with the other peptide. In contrast, when rat placental membrane is used with IGF-II tracer, a high specificity for IGF-II has been reported. Daughaday et al., J. Clin. Endocrinol. Metab., 53: 289-294 (1981). A IGF-II RRA has been established using ovine placental membranes with a specificity comparable to that of human placental IGF-II RRA and a sensitivity somewhat better than that reported for the rat placental membrane assay. The rat liver plasma membrane RRA preferentially measures IGF-II-related peptides. Although the RRA provides an advance over bioassays in ease of performance and reproducibility, specificity and interference by binding proteins remain problematic.
In the circulation, in other body fluids, and in media conditioned by cultured cells, the somatomedins are bound to specific high-affinity carrier proteins that have been implicated as modulators of IGF actions. The history of IGF binding proteins (BPs) dates back to 1984 when the existence of specific somatomedin carrier proteins in serum was first shown. Hintz, Clin. Endocrinol. Metab., 13: 31-42 (1984). Three distinct IGF BPs have now been cloned and sequenced, and in addition, several other, not yet thoroughly characterized, BP species have been identified in various tissues. On the basis of the sequences it became evident that many of the previously recognized BPs, known by different names, were in fact the same, falling into three classes of cloned BPs. To clarify the present status of these BPs, the Workshop on IGF Binding Proteins, held in Vancouver, Canada, June 1989, proposed the names IGFBP-1, IGFBP-2, and IGFBP-3 for the binding proteins with defined sequences. Ballard et al., Acta Endocrinol. (Copenh). 121: 751-752 (1989). The consensus at the Workshop was that other, incompletely characterized IGFBPs be referred to by size and origin until sequenced.
Amniotic fluid was the first source from which IGFBP-1 was detected. Chochinov et al., J. Clin. Endocrinol. Metab., 44: 902-908 (1977). The protein has been purified also from tissue extract of fetal and maternal placenta and named placental protein. Kiostinen et al., Endocrinology, 118: 1375-1378 (1986). The mature protein contains 234 amino acids, predicting a molecular mass of 25.3 kD. Lee et al., Mol. Endocrinol., 2: 404-411 (1988); WO 89/09792 published 19 Oct. 1989. IGFBP-1 migrates on SDS-PAGE at 28-35 kD depending on the stage of reduction. IGFBP-1 is a minor binding protein in serum and contains the unsaturated serum IGF-binding sites. Serum levels are inversely dependent on insulin and have a marked diurnal variation, the levels being highest early in the morning. These levels increase in pregnancy up to several hundred .mu.g/l, and amniotic fluid levels are up to 1000-fold higher than those in serum.
Carrier proteins of the IGFBP-2 class have been isolated from human fetal liver and rat and bovine cell lines. Binkert et al., EMBO J., 8: 2497-2502 (1989). In humans, the mature form contains 289 amino acids and has an apparent molecular mass of 31-40 kD, depending on the stage of reduction on SDS-PAGE. In humans high IGFBP-2 levels have been found in the cerebrospinal fluid. The abundance of this protein in fetal tissue suggests that it has a role in regulating development. IGFBP-2 preferentially binds IGF-II. The majority of serum IGFs are bound to a binding protein composed of two parts forming a complex of molecular mass 125-150 kD. IGFBP-3 is the IGF binding subunit (.beta.-subunit) in this complex. Baxter and Martin, Proc. Natl. Acad. Sci. USA, 86: 6898-6902 (1989). It is an acid-stable glycoprotein appearing on SDSPAGE as a major and minor band, corresponding to 53 and 47 kD, respectively. The other components in the complex are the acid-labile, non-IGF-binding subunit (.alpha.-subunit) with a molecular mass of 84-86 kD [Baxter, WO 90/0569], and IGF-I or IGF-II (.gamma.-subunit). Sequencing of the cloned cDNA for IGFBP-3 (previously known as IGFBP-53) predicts a molecular mass of 28.7 kD for the non-glycosylated protein and reveals that IGFBP-3 shares 33 % sequence identity with IGFBP-1. Wood et al., Mol. Endocrinol., 2: 1176-1185 (1988); Ep 375,438 published 27 Jun. 1990; WO 89/09268 published 5 Oct. 1989.
The levels of IGFBP in adult serum have been found to reflect the growth hormone (GH) status of individuals who are either GH-deficient or acromegalic. Thus, high levels of IGFBP-3 correlate with high levels of GH. Martin and Baxter, J. Clin. Endo. and Metabol., 61: 799-801 (1985). Under normal conditions about 95-98% of the IGF-I in human plasma is bound to the IGFBPs. Studies on size-fractionated human serum, subjected to IGF-I RIA after extraction of each fraction to remove binding activity, have indicated that 72% of the endogenous peptide is associated with the 150-kD fraction and 25% with the 50-kD fraction. Daughaday et al., J. Clin. Endocrinol. Metab., 55: 916-921 (1982).
After acid treatment of the serum to remove endogenous ligands, the IGFBPs (in purified form) have been used as a source of binding sites in competitive protein binding assays for IGF-I. Schalch et al., J. Clin. Endocrinol. Metab., 46: 664-671 (1978); Zapf et al., Clin. Chem., 23: 677-682 (1977). The acid treatment consists of chromatography of serum in dilute acetic acid on gel permeation columns. Binding activity has been reported to emerge between 60 and 80% or between 29 and 46% of the bed volume on Sephadex G-200 depending on the laboratory. To separate IGF-I from endogenous binding protein in samples for assay, each specimen is passed through a Sephadex G-50 column under acid conditions. Fractions emerging between about 50 and 80% of the bed volume are then pooled and assayed. Incubations are for 60-90 minutes at room temperature, after which bound and free ligand are separated using charcoal.
Using IGFBPs from human serum, this technique predominately measures IGF-II. Tracer IGF-II is reported to show three times higher specific binding than tracer IGF-I, and unlabeled IGF-II is more potent in displacing either tracer from binding sites than IGF-I. Zapf et al., Eur. J. Biochem., 87: 285-296 (1978). See also Rechler et al., Endocrinology, 107: 1451-1459 (1980). Activity measured by a method that has relative specificity for IGF-II shows relatively poor dependence on GH status. However, a competitive binding assay has been described utilizing a IGFBP released by rat liver explants in organ culture, which preferentially measures IGF-I, although with 50% IGF-II cross-reactivity. Binoux et al., J. Clin. Endocrinol. Metab., 59: 453-462 (1984). An RIA based on IGFBP-2 is described by Drop et al., J. Clin. Endocrin and Metabol., 59: 899-907 (1984).
Since the RRA and IGFBP assays have limitations resulting from specificity and interference by IGFBPs, they are not the preferred tools for making diagnoses or decisions regarding therapy. Therefore, radioimmunoassays (RIA) have been developed. The first successful RIA method for IGF-I was described by Furlanetto et al., J. Clin. Invest., 60: 648-657 (1977), who was able to raise high-affinity antisera with great specificity for IGF-I. With this antisera, the assay shows half-maximal displacement of tracer by about 0.1 ng of IGF-I, with somatomedin-A less than 5% as reactive, and even lower cross-reactions by human and rat IGF-II. RIAs have been developed subsequently by Bala and Bhaumick, J. Clin. Endocrin. and Metabol., 49: 770-777 (1979) and Zapf et al., J. Clin. Invest., 68: 1321-1330 (1981). Moreover, two antisera have been raised against a synthetic carboxy-terminal octapeptide and the 12 amino acid C-peptide region, the two regions of greatest difference between IGF-I and IGF-II, and between the IGF peptides and proinsulin. The one raised against the carboxy-terminal region, when used with intact IGF-I tracer, had very low cross-reactivity with rat and human IGF-II [Hintz et al., J. Clin. Endocrinol. Metab., 51: 672-673 (1980)], while the C-peptide antibody showed no IGF-II cross-reactivity even at 1000 ng/ml. Hintz et al., J. Clin. Endocrinol. Metab., 50: 405-407 (1980). See also Hintz et al., J. Clin. Endocrinol. Metab., 55: 927-930 (1982). For other assays, see, e.g., Hall et al., J. Clin. Endocrinol. Metab., 48: 271-278 (1979); EP 292,656 published 30 Nov. 1988.
Specific assays for human and rat IGF-II have also been developed. See, e.g., Zapf et al., Metab. Clin. Exp., 27: 1803-1828 (1978); Zapf et al., Acta Endocrinol., 95: 505 (1980); Zapf et al., J. Clin. Invest., supra; Hintz and Liu, J. Clin. Endocrinol. Metabol., 54: 442-446 (1982); Moses et al., Eur. J. Biochem., 103: 401-408 (1980). The assays for IGF-II have shown that normally there is a higher content of IGF-II than IGF-I in plasma. This is confirmed by the specific RRA for IGF-II utilizing rat placental membranes.
Also, monoclonal antibodies suitable for IGF-I RIA have been developed. See, e.g., Baxter et al., J. Clin. Endocrinol. Metab., 54: 474-476 (1982); Laubli et al., FEBS Lett., 149: 109-112 (1982).
The RIAs represent an advance in specificity over the RRAs and bioassays, being more specific to IGF-I than IGF-II, and in general are more sensitive than the other assays. They have been used in a variety of clinical disorders and appear to reflect GH status reliably.
The major problem with measuring IGF-I or -II by RIA is interference by IGFBPs. This interference takes a variety of forms. If the IGFBPs bind I-labeled IGF-I preferentially, a false elevation of the plasma IGF-I concentration will occur. If, however, they interfere with the binding of unlabeled IGF-I to antibody, IGF-I levels will be underestimated. Where a monoclonal antibody was used, unextracted samples were almost devoid of activity, even when containing high levels of antigen. The traditional means to overcome this problem is to separate IGFBPs from free peptide by acid gel filtration chromatography before IGF determination. Zapf et al., J. Clin. Invest., supra.
Several investigators have attempted to circumvent the need for chromatography by nullifying the effect of the IGFBPs using various chemical treatments. These include incubation of samples with acid, followed by lyophilization [Bala and Bhaumick, supra] or neutralization [Binoux et al., Acta Endocrinologica. 99: 422-430 (1982)]; or the exposure to heparin in the RIA buffer [Furlanetto, J. Clin. Endocrin. and Metabol., 54: 1084-1086 (1982)]. These acid treatment procedures destroy the acid-labile component of the IGFBP complex, but not the acid-stable component.
To avoid these difficulties, a direct RIA that does not depend on prior chemical treatment of samples has been developed in which the plasma is collected in EDTA and the assay is performed in the presence of protamine using an antibody of sufficiently high affinity. Chatelain et al., J. Clin. Endocrin. and Metab., 56: 376-383 (1983). One disadvantage of this method is that only a fraction of the total IGF-I content of plasma is measured. Further, due to the reduced affinity of the IGFBP for IGF-I induced by EDTA, the value obtained will vary greatly depending on the amount of time that the sample has incubated at temperatures greater than 4.degree. C.
For those RIAs where extraction is used, a non-equilibrium incubation protocol in which samples and standard are incubated with antibody for several days before addition of tracer, may be required to ensure parallelism between unextracted serum samples and purified samples. However, this method may allow the detection of as little as 20% of the total IGF-I in human serum and the proportion detected may vary depending upon the GH status of the patient. Also, the use of glass or polystyrene tubes, the presence of heparin, and the length of time the sample has stood at neutral or low pH all influence the ability of the antibody to detect antigen in the presence of IGFBPs.
An assay method for free IGF-I levels utilizing IGFBP-3 antibody to separate the IGF-I-BP complex from the free IGF-I in fluids and measuring free IGF-I by RIA is suggested by EP 375,438 published 27 Jun. 1990, page 8.
IGF samples can also be prepared for assay by acid-ethanol extraction. Baxter et al., Clin. Chem., 28: 488-495 (1982); Daughaday et al., J. Clin. Endocrinol. Metab., 781-788 (1980). The acid-ethanol extraction procedure has only been validated for human serum, and may not be fully effective on other sample types, or in different species. Thus, it may not remove all binding activity from human seminal plasma or from conditioned hepatocyte culture media. In the latter case the problem was overcome by adapting the acid gel chromatography technique to a high-pressure liquid chromatography with automated sample injection. Scott et al., Endocrinology, 116: 1094-1101 (1985).
As to measuring free IGF-I in the plasma, one of the traditional methods is to place the serum on a gel filtration column under neutral conditions, cut out fractions representing each peak obtained, and measure the free IGF-I level in each fraction by RIA. Unfortunately, this method is not sensitive due to extensive dilution, results in varying recovery levels, is subject to interference from some IGFBPs that overlap in peaks, and is time-consuming because only one sample at a time can be measured.
To overcome these problems, one method is to precipitate the IGFBPs and complexes and measure free IGF-I in the supernatant. Precipitation methods include ammonium sulfate or polyethylene glycol precipitation, or immunoprecipitation. However, precipitation may be incomplete or the free IGF-I may precipitate. Another method involves ultrafiltration of the serum [Daughaday et al., J. Clin. Endocrin and Metab., 55: 916-921 (1982)] or equilibrium dialysis, which requires a long period of time and is not practical for large numbers of samples.
Another technique involves passing the serum through a small cartridge (e.g., Sep-Pak sold by Millipore) that retains the free IGF-I while allowing the bound IGF-I to flow through. Hizuka et al., 71st Endocrine Society Annual Meeting, Program Abstracts, 1989, Abst. #1020. The column is washed and eluted with organic solvent or acid to recover the free IGF-I. The resulting fraction is then concentrated (dried and lyophilized), reconstituted, and subjected to RIA. This technique suffers from variability in cartridges and recovery, the need for lyophilization or concentration, and impracticality. While this method allows analysis of more samples than the other methods, still only 30 to 40 samples a day can be processed.
Detection of other polypeptide hormones that circulate bound to one or more BPs has also been hampered by the above considerations. A well characterized such BP is the high-affinity growth hormone binding protein (GHBP) constituting the extracellular domain of the GH receptor that circulates in blood and functions as a GHBP in several species [Ymer and Herington, Mol. Cell. Endocrino., 41: 153 (1985); Smith and Talamantes, Endocrinology, 123: 1489-1494 (1988); Emtner and Roos, Acta Endocrinologica (Copenh.). 122: 296-302 (1990)], including man. Baumann et al., J. Clin. Endocrinol. Metab., 62: 134-141 (1986): EP 366,710 published 9 May 1990; Herington et al., J. Clin. Invest., 77: 1817-1823 (1986): Leung et al., Nature, 330: 537-543 (1987). A second BP with lower affinity for GH has also been described that appears to be structurally unrelated to the GH receptor. Baumann and Shaw, J. Clin. Endocrinol. Metab., 70: 680-686 (1990). Assays for, inter alia, GH are described e.g., in JP 86000941 published 13 Jan. 1986; JP 55037949 published 17 Mar. 1980; JP 85004423 published 4 Feb. 1985; DD 276,532 published 28 Feb. 1990; Ep 58,486 published 25 Aug. 1982; and U.S. Pat. Nos. 4,185,084 and 3,555,143.
Specific binding pairs (SBP), e.g., antigen-antibody, ligand-receptor, hormone-receptor, and lectin-oligosaccharide pairs, are discussed in U.S. Pat. No. 4,956,302. Assays of whole blood to detect a SBP member wherein a first member is immobilized prior to contact with the blood and the second member is detected through competition with labeled second member are reported in U.S. Pat. No. 4,594,327. A method to reduce interfering substances in assays of a SBP member wherein a SBP member is labeled and a receptor is used to capture both labeled and unlabeled SBP member is found in U.S. Pat. No. 4,737,456.
Further, free small analytes in samples are determined by absorbing at least a portion of the analyte with an insolubilized "receptor," washing the insoluble receptor, adding labeled sample analyte or receptor, incubating, washing the insoluble phase free of soluble phase, and determining the label, as described in U.S. Pat. No. 4,292,296. This method, along with the methods described by U.S. Pat. Nos. 4,680,275 and 4,704,355, utilizes antibodies or antigens as the receptor for the insoluble phase. Other solid-phase systems for ligand assays that involve determination of any analytes, not limited to those that circulate in complexed form, are described in U.S. Pat. Nos. 4,786,606 and 4,279,885. A method for detecting an inhibitor of a plasminogen activator wherein the activator may be immobilized is described in U.S. Pat. No. 4,791,068.
Other assay systems involving immobilized phases include those described by U.S. Pat. No. 4,015,939 (intermediate base anion exchange resin), U.S. Pat. No. 4,594,327 (assay for whole blood samples), U.S. Pat. No. 4,333,918 (assay for vitamin B12), U.S. Pat. No. 4,251,360 (utilizes tagged component), U.S. Pat. No. 4,219,335 (immunochemical testing assay), U.S. Pat. No. 4,005,187 (contacting two solutes in the fluid), U.S. Pat. No. 4,921,808 (assaying follicle stimulating hormone), U.S. Pat. No. 4,886,761 (polysilicon binding assay), U.S. Pat. No. 4,742,011 (assay device), and U.S. Pat. No. 4,943,522 (lateral flow assay), EP 166,623 (utilizes immunological reaction); JP 1098969 (immobilizing a soluble receptor); JP 62098260 (kit of enzyme-labeled antibody or antigen, protein, sheet to bind protein, substrate for the enzyme, and buffer liquid); and JP 85004423 (immobilizing receptor).
An object of the present invention is to provide an assay for free IGF-I, IGF-II, and GH in a body fluid that normally contains a proportion of IGF-I, IGF-II, and/or GH complexed with one or more binding proteins, which assay is not chromatographic and does not utilize an antibody as a capture reagent.
Another object is to provide an assay for such proteins that provides a number of advantages relating to ease and speed of analysis, reliability, sensitivity, selectivity, and precision.
These and other objects will be apparent to one of ordinary skill in the art.